Thin layer chromatography has acquired great importance as a relatively inexpensive and easily applied analytical tool in the modern laboratory. Such chromatography employs a finely divided adsorbent, commonly silica gel or alumina, bonded in a thin, usually uniform or flat, layer on a supportive substrate, usually glass, to form a thin layer chromatographic plate, as it is called in the trade.
In use, the sample mixture to be resolved, usually one drop of solution, is placed on the adsorbent near one edge of the plate. After drying, an eluting solvent is applied to the same edge of the plate, generally by setting the plate upright in a reservoir having a small amount of eluting solvent which then rises up the plate by capillary action. The components of the sample mixture move with the eluting solvent at rates which are inversely proportional to the strength of their attraction to the adsorbent, relative to the solubility in the solvent. Thus, on development, as it is called, the components of the mixture which, for this and other reasons, move at different rates, are distributed along the solvent path. At this point, the plate is said to be developed.
The position of a separated component on the developed plate is established by means of the intrinsic color of the component, by means of color forming reagents sprayed on the dried plate, by optical means such as ultraviolet light irradiation, or by other means depending on the properties of the components being separated.
The separated components are identified according to the distances through which they have moved, expressed as a fraction of the distance through which the solvent front has moved. These fractions are the so-called R.sub.f values and are, under standard conditions, characteristic of each component.
Commonly used plates are flat, that is the thickness of the adsorbent layer is the same over the whole area of the plate. Typically, adsorbent layers for analytical purposes are 100 to 250 .mu.m thick. Such plates, for example those using silica gel, can accept sample solutions ranging from 1 to 20 .mu.l, the solutions containing from about 1 to about 10 .mu.g of solute.
Occasionally there is need to treat larger samples, for example to provide purified materials for further work (so-called preparative chromatography), or to separate components present in tiny amounts from relatively large amounts of other materials. Plates having thicker adsorbent layers up to about 2,000 .mu.m are commercially available for such purposes. However, such plates often provide unsatisfactory resolution. There appear to be at least two reasons for this. Firstly, as is obvious, large sample volumes, even on thick absorbent layers, will produce large sample spots wherein the components at the starting line are distributed over the area of the spot. It follows, of course, that a diffuse starting spot (or line as is generally used in preparative chromatography) will produce a diffuse distribution of component spots (or bands) on development and components having similar R.sub.f values will not be resolved. The other principal reason is related to the fact that in thicker adsorbent layers the components are carried forward often in tear drop shape due to so-called wall effects, the component front generally lagging behind at the adsorbent-air interface. At the adsorbent-support interface, the component front either lags behind or moves ahead, depending on the wetting characteristics of the component with respect to the support. In general, the faster a component moves the more pronounced the wall effects. Finally, the slower drying of thicker adsorbent layers after development also permits greater outward diffusion of separated components from the spot or band during the drying process.
Now available in the trade is a type of chromatographic plate comprising a so-called preconcentrating zone which overcomes to a degree problems associated with diffuse sample spots or lines at the plate starting line. The preconcentrating zone comprises a granular nonabsorbent such as inactive kieselguhr or glass powder placed adjacent to the regular adsorbent along the starting edge of the plate where sample is applied. When eluting solvent is applied to the plate, all components, being unadsorbed in the preconcentrating zone, move rapidly out of the zone with the solvent front until they meet the regular adsorbent and are slowed. The result is that all components are concentrated on a thin starting line at the bottom of the adsorbent.
Other stratigems intended to improve the resolution of thin layer chromatography, usually with respect to particular components, have been disclosed. For example, Abbott et al.sup.(1) in Chemistry and Industry (London) 481 (1964) teach the use of what they call a "wedge layer plate". In this plate, the thickness of the adsorbent layer tapers from 2,000 .mu.m at the starting edge to 100 .mu.m at the opposite edge. Abbott et al teach the use of the thicker adsorbent at the origin to remove coextracted chlorophylls and carotenes (polar materials), i.e., to "clean up" the mixture as the authors say, in order better to resolve large R.sub.f value chlorophenoxy acid herbicides in the thinner sections.
In Chemistry and Industry 310 (1965), Abbott et al.sup.(2) teach the use of a wedge layer plate wherein the thick section of the wedge is formed from highly active material while the remainder of the layer is of less retentive composition.
Analogously, Bazan Jr. et al in Journal of Lipid Research 11, 42 (1970) teach similar wedge layer plate resolution in the thin section of trace amounts of free fatty acids in complex lipid extracts.
The wedge layer plates of the art have in common the notion of placing the largest amount of adsorbent, or the greatest adsorptivity, near the starting edge and lesser amounts in the region of the opposite edge, where the components of particular interest are to be resolved.